Cloning and Sequence of Nicotinic Acetylcholine Receptor α Subunit from Chilo suppressalis

Nicotinic acetylcholine receptors (nAChRs) play a significant role in excitatory synaptic transmission in insects and are the target for chloronicotinyl and nereistoxin insecticides.In recent years,Chilo suppressalis,an economically important pest of rice,developed high resistance against monosultap,a nereistoxin insecticide acting on nAChR.In order to reveal the hypothesized target insensitive mechanism,studies on the molecular property of nAChR from Chilo suppressalis are required.In this study,the full length cDNA of nAChR α subunit from this pest was cloned by RT-PCR.Sequence analysis shows that it is a novel nAChR α subunit,which was named as Cs α 1(Genbank accession No.AF418987).It contains 1?997?bp nucleotides and involves an open reading frame (ORF) encoding a mature protein of 509 amino acids excluding a signal peptide of 24 amino acids.The deduced amino acid sequence was 52%-94% identical to the reported insect nAChR genes.

Cloning,Sequencing and Molecular Phylogenetic Analysis of the Mitochondrial Cytochrome Oxidase Ⅰ Gene of Chilo suppressalis

[Objective] The research aimed at cloning and analyzing mitochondrial cytochrome oxidase I gene（cox 1）of C.suppressalis.[Method] The mitochondrial cox 1 gene of C.suppressalis was cloned with PCR method and sequenced.Then,cox1 sequences of other 21 Lepidopteran species were obtained by blasting the GenBank with cox 1 gene sequence of C.suppressalis.Finally,homology comparison and molecular phylogenitic analysis among the 22 Lepidopteran species were conducted.[Result] The open reading frame of cox 1 gene from C.suppressalis contained 1 531 nucleotides encoding a putative protein of 510 amino acids.The cox1 gene used a start codon CGA,and an incomplete termination codon composed of only T.Based on the amino acid sequences of cox 1,the molecular phylogenetic tree of Lepidoptera was reconstructed using the maximum likelihood（ML）method.The molecular phylogenetic tree was similar to the morphological phylogenetic tree mainly,but also showed some differences.[Conclusion] The result will provide reference for further research on expression and application of cox 1 gene.

Effect of Rapid Cold Hardening on the Cold Tolerance of the Larvae of the Rice Stem Borer,Chilo suppressalis(Walker)

The effects of rapid cold hardening （RCH） on the cold tolerance of the last instar larvae of Chilo suppressalis （Walker） were evaluated for the first time. The discriminating temperature, induction, detection, duration and extent of RCH of the larvae in the laboratory were tested, and the supercooling points （SCPs） and the contents of water and lipid of the larvae after RCH treatment were determined, respectively. The results showed that the discriminating temperature of the larvae was about -21℃. Mean survival rates of the larvae which exposed to either 0 or 5℃ for 2 and 4 h before exposure to the discriminating temperature for 2 h were significantly higher than those of the control groups （P 〈 0.05）. Moreover, the highest survival rate appeared in the larvae after 0℃ for 4 h treatment. The protection against low temperature gained by RCH at 0℃ for 4 h was rapidly lost on return to 28℃. Mean survival rates of RCH larvae were significantly higher than those of non-acclimated （NACC） larvae and acclimation （ACC） larvae when they were exposed to the discriminating temperature for 2 or 4 h （P〈 0.05）. Moreover, the rates of NACC, ACC, RCH and ACC ＋ RCH larvae from 2 to 6 h to the discriminating temperature resulted in a significant decline. The values of SCPs and the contents of lipid of the larvae which exposed to either 0 or 5℃ for 2 and 4 h showed no significant difference at 0.05 level compared to those of the control groups. But the contents of water in the larvae were obviously decreased. Therefore, it could be concluded that RCH could enhance cold tolerance and affect partly physiological and biochemical components of the larvae of C. suppressalis, but the underlying mechanisms needs to be further explored.

Evaluation of Lethal Effects of Chlorantraniliprole on Chilo suppressalis and Its Larval Parasitoid, Cotesia chilonis

Chlorantraniliprole was introduced into China from 2008 as a novel insecticide to control the rice stem borer, Chilo suppressalis （Walker）. Cotesia chilonis （Matsumura） is the major parasitoid of C. suppressalis. We collected seven populations of C. suppressalis and two populations of C. chilonis in different locations in China in 2009 to investigate the lethal effects of chlorantraniliprole on them. The populations of C. suppressalis from different regions showed various LD50 values ranging from 2.00 to 18.70 ng per larva. Chlorantraniliprole has negligible acute contact toxicity （LC50500 mg L-1） to C. chilonis and its oral toxicity is also much lower than that of fipronil （2 800-fold difference in LC50）. The results indicated that chlorantraniliprole is a good alterative in rice integrated pest management （IPM） programs. The susceptibility data of C. suppressalis will be useful for monitoring resistance levels in future.

Electrophysiological responses of the rice striped stem borer Chilo suppressalis to volatiles of the trap plant vetiver grass (Vetiveria zizanioides L.)

Vetiver grass （Vetiveria zizanioides L.） was previously found to effectively attract female adults of Chilo suppressalis （Walker）, an important pest of rice. To determine the volatile compounds involved in this attraction, electroantennography （EAG） responses to seven synthetic volatiles released from vetiver plants were examined. Our results indicated that the responses of C. suppressalis adult antennae to the different compounds varied widely. The compounds elicited strong EAG responses in female antennae were subsequently selected for further EAG response tests, namely, caryophyllene, β-ocimene, linalool and a-pinene. EAG responses to a combination of these four compounds did not differ significantly from the individual compounds. However, pair combination tests indicated that 0.01μg μL-1linalool and 50 μg μL-1 α-pinene, 50 μgμL-1 caryophyllene and 0.01μg μL-1 linalool, 0.01 μg μL-1 13-ocimene and 0.01 μg μL-1 linalool, and 0.01μg μL-1 β-ocimene and 50 μg μL-1 caryophyllene elicited significantly greater EAG responses in 3-day female moths compared to the 1-day female. These compound combinations and the corresponding ratios are probably playing an important role in attracting female adults of C. suppressalis to the vetiver grass.

Preliminary Study on Resistance of the Rice Stem Borer (Chilo Suppressalis) to Fipronil

By means of topical application, fipronil resistance was surveyed in the rice stem borer, Chilo suppressalis, from 12 diferent areas in east China from 2001 to 2004. The rice stem borers in most regions of Jiangsu and Anhui were still susceptible to fipronil. But in Wuxi, Jiangsu Province and Cixi, Zhejiang Province, their sensitivity became decreased （resistance ratios were 3.1 and 3.6, respectively）, and the medium level of resistance （resistance ratio was 21.2） was found in Cangnan, Zhejiang Province. So, it was still at the early stage for fipronil resistance development in this pest. Synergism experiments showed that piperonyl butoxide（PBO） had a little effect on both susceptible and resistant borers （synergism ratios were 1.1-1.2）. Though triphenyl phosphate （TPP） and diethyl meleate （DEM） had no effect on the susceptible borers, they had significant synergism on fipronil in the resistant population to fipronil （synergism ratios were 1.8 and 1.6, respectively）, indicating esterase and glutathion S-transferase may be involved in the resistance mechanism. Bioassay with currently used insecticides indicated that triazophos （because of high resistance）, trichlorphon and acephate had very low toxicity to resistant borers. But diazinon, pyridaphenthion, decamethrin and avermeatin showed high toxicity and had no cross resistance to fipronil, which could be considered as substitute insecticides in the resistance managment.

Population Genetic Analysis of the Rice Stem Borer, Chilo suppressalis, in the South China

Genetic variation and patterns of genetic differentiation of the rice stem borer, Chilo suppressalis （Lepidoptera： Pyralidae）, from the South China were analyzed using 6 microsatellite markers and two partial mtDNA （cox1 and cox2） regions. All of the 6 microsatellite loci were polymorphic in the studied seven populations. The allelic richness per population ranged between 5.67 and 14.00, and average H E and H O values were 0.6246-0.8329 and 0.2634-0.6061, respectively. As the mitochondrial genome is a single genetic locus, we only present results for the concatenated data set （cox1 plus cox2 gene sequences, 513 bp）. The concatenated data showed high level of genetic diversity and there are 23 variable polymorphic sites among the 513 sites in concatenated data. Nearly all of （20 of 21） pairwise F ST comparisons among populations showed genetic differentiation with moderate to high pairwise F ST values based on microsatellite markers. However, for the mtDNA data, most of the seven populations did not show significant differentiation with other populations. The differences of population differentiation obtained with the two different genetic markers could be mainly attributed to the different mutation rates of microsatellite and mtDNA. There was not genetic structure existed in these studied populations based on microsatellite loci and mtDNA data. The analysis based on network, mismatch distribution, Tajima＇s D and F S indicated that the studied populations were from the recent same ancestor or the same refuge and followed by a sudden demographic expansion condition.

Suppression of Rice Stem Borer, Chilo suppressalis by Mass Trapping Using Synthetic Sex Pheromone in Paddy Field

Suppressing effects of mass trapping using synthetic sex pheromone with main active ingredients of Z-11-hexadecenal, Z-13-octadecenal and Z-9-hexadecenal on the rice stem borer, Chilo suppressalis were investigated during the flight periods of the overwintering and 1st generation in the paddy area of 60 ha in 1999. Population density of C. suppressalis in the pheromone-treated fields was lower than that in control. The egg masses decreased by 74.39% and population size of adult males of the 1st generation decreased by 61.64% in the treated fields as compared to control. Meanwhile, the damage by C. suppressalis larvae was significantly lower in the treated fields than control (t-test, P=0.05). Percentages of brownish leaf sheath, dead heart, and white head in the treated fields were decreased by 70.90%, 57.01% and 44.30%, respectively in contrast to control. The present study demonstrated that mass trapping to C. suppressalis using synthetic sex pheromone shows great potential as an alternative measure in an environment-friendly pest management and at the same challenging the insecticide use that has some environment impacts.

Effects of High-Quality Aromatic Rice Varieties on the Fitness of the Striped Stem Borer, Chilo suppressalis (Walker) in Central China

The striped stem borer （SSB）, Chilo suppressalis （Walker） （Lepidoptera： Pyralidae）, is a major pest of rice in China. Variation in host-plant quality may affect the body size of herbivorous insects, which in turn, can determine their life-history parameters such as survival, fecundity and fitness. In this study, we tested the effects of high-quality aromatic rice varieties on the fitness of SSB in China. Results showed that 1st instar larvae had higher penetrating rates and survival rates on the high-quality aromatic rice varieties （EX-1 and WX-988） than that on the non-aromatic rice varieties （EZ-5 and LYP-9）. In addition, shorter developmental periods, greater female pupal weights and higher of other life-history parameters （hatching rate, pupation rate, eclosion rate and increase index） were found on the high-quality aromatic rice varieties, although only female pupal weight showed a significant difference between the two varieties. The highest dead heart rate was found on the aromatic rice variety of EX-1. These results indicate that SSB sustains a lower fitness cost when consuming the high-quality aromatic rice varieties （EX-1 and WX-988） than on the non-aromatic rice varieties （EZ-5 and LYP-9） in Central China.

Characterization of T-complex polypeptide 1 (TCP-1) from the Chilo suppressalis HSP60 family and its expression in response to temperature stress

Many proteins require assistance from molecular chaperones at various stages to attain correctly folded states and functional conformations during protein synthesis. In this study, the gene encoding T-complex polypeptide 1（TCP-1）, which belongs to the heat shock protein 60（HSP60） family, was isolated and characterized from the rice stem borer, Chilo suppressalis, by RACE and q PCR, respectively. The full-length c DNA of Tcp-1 was 2 144 bp and encoded a 1 635-bp ORF; the deduced translational product contained 545 amino acids with 5′-and 3′-UTRs and an isoelectric point of 5.29. Cluster analysis confirmed that the deduced amino acid sequence shared high identity（60–99%） with TCP-1 from other insects. To investigate Tcp-1 expression in response to abiotic stress, q PCR was used to analyze expression levels of Tcp-1 m RNA in C. suppressalis larvae exposed to temperatures ranging from –11 to 43°C. With respect to heat shock, Tcp-1 expression was higher than the control after a 2-h exposure to 30 and 36°C and declined at 39 and 43°C. Difference in Tcp-1 expression was observed at temperatures ranging from –11 to 27°C. q PCR analyses revealed that Tcp-1 expression was the highest in hindgut tissue as compared to heads, epidermis, fat body, foregut, midgut, and malpighian tubules. Our results indicated that Tcp-1 expression was differentially expressed in C. suppressalis tissues, and was impacted by temperature stress.

Cloning,Tissue Distribution,and Transmembrane Orientation of the Olfactory Co-Receptor Orco from Two Important Lepidopteran Rice Pests,the Leaffolder(Cnaphalocrocis medinalis) and the Striped Stem Borer(Chilo suppressalis)

In insects,the sense of smell is mainly mediated by olfactory receptors（Ors）.Olfactory co-receptor（Orco）,which is coexpressed with the Ors in almost all olfactory receptor neurons（ORNs）,is demonstrated to be an essential component in the insect olfactory system.It can be potential target for developing novel olfactory-disruption strategy to control insect pests.In this study,two full-length cDNA sequences encoding Orcos（CmedOrco and ChsupOrco） were cloned from two Lepidopteran rice pests,the rice leaffolder,Cnaphalocrocis medinalis and the rice striped stem borer,Chilo suppressalis.The amino acid sequences of CmedOrco and ChsupOrco showed high similarity to the previously identified Orcos from other insect species. Bioinformatic prediction and cellular immunofluorescence indicated that CmedOrco and ChsupOrco were both seven-transmembrane proteins with intracellular N-termini and extracellular C-termini.mRNA expression levels of the two Orcos were much higher in male and female antennae than those in non-olfactory tissues,and the ChsupOrco transcripts reached a peak level in adults compared to other life stages.Our results provide a foundation from which it will be possible to elucidate the roles of Orco in moth olfaction and for the development of environment-friendly management strategies of these two rice insect pests.

The insecticide resistance in stripped stem borer,Chilo suppressalis(Walker)

The stripped stem borer (SSB),Chilo suppressalis (Walker) is one ofthe major insect pests of rice in Chi-na. Chemical control has been acommon practice in SSB managementsince 1950s. Insecticides used in-cluded BHC before 1983; organophos-

Identification of Bt-transgenic Rice Plants for Resistance to Stripe Stem Borer (Chilo suppressalis) and Genetic Analysis of the Transgenes

Resistance to herbicide Basta was used to identify Bt-transgenic rice plants and the progenies of crosses between the Bt-transgenic rice and the rice varieties cultivated in the Huang Huai area of China. The results demonstrated that the Basta-positive rice plants were highly resistant to stripe stem borer (Chilo sup-pressalis) both in the laboratory and field tests. Both cryIA (b) and bar genes were expressed and co-inherited in both self ing and crossing progenies. Mendelian segregation of the marker gene bar was observed in F2 and BC1 progenies. The results implicates that it is possible to transfer cryIA (b) gene into other cultivated varieties through crosses and back crosses.

A New and Simple Method for Collecting Eggs of the Striped Stemborer,Chilo suppressalis

The method for collecting eggs of the striped stem borer, Chilo suppressalis using plastic bags was studied in comparison with using caged rice plants. There was no significant difference in fecundity of C. suppressalis at 279 eggs/moth and in percentage of hatched eggs at 95% between in plastic bags and on rice plants. More egg masses were collected on plastic bags than on rice plants, whereas more smaller egg masses (less than 100 eggs per mass) in plastic bags than on rice plants. The advantages in collecting eggs of C. suppressalis and other insects by using plastic bags were also discussed.

The Effect and Benefit of Rice Chilo suppressalis Walker Chemical Control Test of Chlorantraniliprole etc.

In this paper, the first generation and second generation of Chilo suppressalis Walker were conducted as the test objects to investigate control of a new pesticide(chlorantraniliprole) with preparation of chlorantraniliprole 20% SC,abamectin 1.8% EC and chlorpyrifos 40% EC to Chilo suppressalis Walker. The results showed that chlorantraniliprole 20% SC, abamectin 1.8% EC and chlorpyrifos40% EC had effective control to Chilo suppressalis Walker for its excellent control efficacy and long persistence period. Seedling protection efficacies were between84.2%-100%, 90.1% and 92.1%, and insecticidal efficacies were between 84.6%-100%,90.7% and 93.8% 36 days after application of chlorantraniliprole 20% SC in treatments with abamectin 1.8% EC and chlorpyrifos 40% EC for first generation Chilo suppressalis Walker control. The dead panicles were between 0.2%-1.4%, 0.8%and 0.7%, pest plant rate control in 0.4%-2.2%, 1.1% and 0.9%, and the rates of damaged rice plants were between 82.4%-97.6%, 90.2%-91.2% and 91.4%-92.8%,30 days after application of chlorantraniliprole 20% SC, abamectin 1.8% EC and chlorpyrifos 40% EC for the second generation Chilo suppressalis Walker control. It can effectively protect seedlings, protect spike, increase grain number and grain weight, which promoting the increase production income effect. The real estate increased by 9.7%-12.3%, 10.9% and 11.2% than water treatment(CK) with a very significant level, while the incomes were higher than 1 719.19-1 998.22, 1 956.52 and 2 057.83 yuan.

Risk assessment,fitness cost,cross-resistance,and mechanism of tetraniliprole resistance in the rice stem borer,Chilo suppressalis

The rice stem borer(RSB),Chilo suppressalis,a notorious rice pest in China,has evolved a high resistance level to commonly used insecticides.Tetraniliprole,a new anthranilic diamide insecticide,effectively controls multiple pests,including RSB.However,the potential resistance risk of RSB to tetraniliprole is still unknown.In this study,the tetraniliprole-selection(Tet-R)strain was obtained through 10 continuous generations of selection with tetraniliprole 30%lethal concentration(LC_(30)).The realized heritability(h^(2))of the Tet-R strain was 0.387,indicating that resistance of RSB to tetraniliprole developed rapidly under the continuous selection of tetraniliprole.The Tet-R strain had a high fitness cost(relative fitness=0.53).We established the susceptibility baseline of RSB to tetraniliprole(lethal concentration at LC_(50)=0.727 mg/L)and investigated the resistance level of 6 field populations to tetraniliprole.All tested strains that had resistance to chlorantraniliprole exhibited moderate-to high-level resistance to tetraniliprole(resistance ratio=27.7-806.8).Detection of ryanodine receptor(RyR)mutations showed that the Y4667C,Y4667D,I4758M,and Y4891F mutations were present in tested RSB field populations.RyR mutations were responsible for the cross-resistance between tetraniliprole and chlorantraniliprole.Further,the clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9-mediated genome-modified flies were used to study the contribution of RyR mutations to tetraniliprole resistance.The order of contribution of a single RyR mutation to tetraniliprole resistance was Y4667D>G4915E>Y4667C~~I4758M>Y4891F.In addition,the I4758M and Y4667C double mutations conferred higher tetraniliprole resistance than single Y4667C mutations.These results can guide resistance management practices for diamides in RSB and other arthropods.

Comparison of three transgenic Bt rice lines for insecticidal protein expression and resistance against a target pest, Chilo suppressalis （Lepidoptera： Crambidae）

Two transgenic rice lines （T2A-1 and T1C-19b） expressing cry2A and crylC genes, respectively, were developed in China, targeting lepidopteran pests including Chilo suppressalis （Walker） （Lepidoptera： Crambidae）. The seasonal expression of Cry proteins in different tissues of the rice lines and their resistance to C. suppressalis were assessed in comparison to a Bt rice line expressing a crylAb/Ac fusion gene, Huahui 1, which has been granted a biosafety certificate. In general, levels of Cry proteins were T2A- 1 〉 Huahui I 〉 T1C-19b among rice lines, and leaf 〉 stern 〉 root among rice tissues. The expression patterns of Cry protein in the rice line plants were similar： higher level at early stages than at later stages with an exception that high Cry 1C level in T 1 C- 19b stems at the maturing stage. The bioassay results revealed that the three transgenic rice lines exhibited significantly high resistance against C. suppressalis larvae throughout the rice growing season. According to Cry protein levels in rice tissues, the raw and corrected mortalities of C. suppressalis caused by each Bt rice line were the highest in the seedling and declined through the j ointing stage with an exception for T1 C-19b providing an excellent performance at the maturing stage. By comparison, T1C-19b exhibited more stable and greater resistance to C. suppressalis larvae than T2A-1, being close to Huahui 1. The results suggest crylC is an ideal Bt gene for plant transformation for lepidopteran pest control, and T1C-19b is a promising Bt rice line for commercial use for tolerating lepidopteran rice pests.

Molecular characterization and expression profiles of nicotinic acetylcholine receptors in the rice striped stem borer, Chilo suppressalis （Lepidoptera： Crambidae）

Nicotinic acetylcholine receptors （nAChRs） are members of the cys-loop ligand- gated ion channel （cysLGIC） superfamily, mediating fast synaptic cholinergic transmission in the central nervous system in insects. Insect nAChRs are the molecular targets of economically important insecticides, such as neonicotinoids and spinosad. Identification and characterization of the nAChR gene family in the rice striped stem borer, Chilo suppressa[is, could provide beneficial information about this important receptor gene family and contribute to the investigation of the molecular modes of insecticide action and resistance for current and future chemical control strategies. We searched our C. suppressalis transcriptome database using Bombyx mori nAChR sequences in local BLAST searches and obtained the putative nAChR subunit complementary DNAs （cDNAs） via reverse transcription polymerase chain reaction （RT-PCR） and rapid amplification of cDNA ends methods. Similar to B. mori, C. suppressalis possesses 12 nAChR subunits, including nine c^-type and three/%type subunits. Quantitative RT-PCR analysis revealed the expression profiles of the nAChR subunits in various tissues, including the brain, subesophageal ganglion, thoracic ganglion, abdominal ganglion, hemocytes, fat body, foregut, midgut, hindgut and Malpighian tubules. Developmental expression analyses showed clear differential expression of nAChR subunits throughout the C. suppressalis life cycle. The identification of nAChR subunits in this study will provide a foundation for investigating the diverse roles played by nAChRs in C. suppressalis and for exploring specific target sites for chemicals that control agricultural pests while sparing beneficial species

Gene cloning and expression of aminopeptidase N and cadherin from midgut of the rice stem borer, Chilo suppressalis

The rice stem borer, Chilo suppressalis Walker is one of the most important insect pests on rice in Asia, north Africa and southern Europe. Transgenic Bt rice has been developed in the laboratory with good resistance to this pest and other Lepidopteran insects, which will provide a possible alternative tool for this pest control. The full-length cDNAs encoding an aminopeptidase N （CsAPN） and a cadherin （CsCad） were cloned from C. suppressalis. CsAPN showed common features of, and high identities to, other insect APNs in its deduced amino acid sequence. Although a full-length cDNA encoding cadherin-like protein has been reported in GenBank, the newly isolated cadherin here （CsCad） showed some differences in its amino acid sequence, especially at the 7th cadherin repeat region （CR7）, which indicated the newly isolated CsCad might be another allele. CsAPN and CsCad were successfully expressed in insect Tn cells, and the blot analysis showed these two proteins could bind Bt toxin CrylAb. The results will provide valuable information for the studies of toxin mode of action and the possible toxin resistance mechanisms in this pest.

Molecular and functional analysis of chitin synthase genes in Chilo suppressalis(Lepidoptera:Crambidae)

The rice stem borer,Chilo suppressalis,has developed a high level of resistance to many of the compounds currently used for control.There is therefore an urgent need to develop novel control methods for C.suppressalis.Insect chitin synthases(CHS)have attracted interest as a potential target for insect pest management.However,to date,CHS have not been characterized in C.suppressalis.Two CHS genes(CsCHSI and CsCHS2)were identified and cloned from C.suppressalis.Two transcript variants were identified for CsCHS1,CsCHSla and CsCHS1b.Spatiotemporal expression profiling showed that both transcripts of CsCHS1 are most highly expressed on the last day of each larval instar stage and show the highest expression levels in the integument.In contrast,CsCHS2 is predominantly expressed during the larval feeding stages and shows the highest expression levels in the midgut.Knockdown of CsCHSI by RNA interference significantly inhibited the molting and pupation of C.suppressalis,and knockdown of CsCHS2 significantly affected growth during the larval stage,but had no significant effect on the pupation.Moreover,knockout of CsCHS1 by CRISPR/Cas9 genome editing severely lowered the hatching rate,larval survivorship,pupation rate,and eclosion rate,but only larval survivorship at the GO generation was lowered after the knockout of CsCHS2.These results demonstrate that CsCHS1 and CsCHS2 play vital roles in the growth and development of C.suppressalis,and so have potential as insecticidal targets for the control of this highly damaging pest.